A general method for the detection of a polynucleotide sequence of interest in a sample comprises:
(a) rendering at least a portion of said polynucleotide sequence of interest in single stranded form; PA1 (b) providing a composition which comprises a first polynucleotide sequence which is substantially complementary and capable of hybridizing to said polynucleotide sequence of interest and which is labeled with a detectable marker; PA1 (c) rendering at least a portion of said composition in substantially single stranded form; PA1 (d) contacting said polynucleotide sequence of interest with said composition under conditions to permit hybridization; and PA1 (e) detecting said polynucleotide sequence of interest by means of said detectable marker. PA1 (a) a first polynucleotide sequence wherein said first polynucleotide sequence is substantially complementary to and capable of hybridizing to said polynucleotide sequence of interest and is labeled with a first detectable marker; PA1 (b) a second polynucleotide sequence wherein said second polynucleotide sequence is not substantially complementary to or substantially identical to said first polynucleotide sequence of interest and is labeled with said first detectable marker; PA1 (c) a third polynucleotide sequence wherein said third polynucleotide sequence is substantially complementary to or identical to said second polynucleotide sequence and is either unlabeled or is labeled with a second detectable marker. PA1 1. a first polynucleotide sequence wherein said first polynucleotide sequence is substantially complementary to and capable of hybridizing to said polynucleotide sequence of interest and is labeled with a first detectable marker; PA1 2. a second polynucleotide sequence wherein said second polynucleotide sequence is not substantially complementary to or substantially identical to said first polynucleotide sequence of interest and is labeled with said first detectable marker; and PA1 3. a third polynucleotide sequence wherein said third polynucleotide sequence is substantially complementary to or identical to said second polynucleotide sequence and is either unlabeled or is labeled with a second detectable marker;
This method is often not useful when: (1) said composition further comprises a second polynucleotide sequence which, either in the same molecule or a separate molecule, is not substantially complementary to said polynucleotide sequence of interest and which is labeled with said detectable marker; and (2) said polynucleotide sequence of interest is potentially contained in a sample that comprises polynucleotide sequences not of interest. When both conditions (1) and (2) are present, any signal detection is ambiguous as to whether said polynucleotide sequence of interest is detected or some polynucleotide sequences not of interest but hybridizable to said labeled second polynucleotide sequence are detected.
As an example, condition (1) presents itself quite naturally when said first polynucleotide sequence is produced by recombinant nucleic acid technology. Recombinant nucleic acid technology allows economic large scale production of said first polynucleotide sequence concommitant with a second polynucleotide sequence which is not substantially complementary to the polynucleotide sequence of interest, the vector sequence in this instance, on the same molecule, i.e. the recombinant molecule. Often, it is easier or more economical to label the entire recombinant molecule than to label exclusively said first polynucleotide sequence. However, this also produces a labeled second polynucleotide sequence, i.e. the vector sequence in this instance, which is not substantially complementary to said polynucleotide of interest.
As another example, condition (1) presents itself when said first polynucleotide sequence is inserted, along with a second polynucleotide sequence not substantially complementary to the polynucleotide sequence of interest, into a vector to form a single recombinant molecule. This is due to the fact that it is difficult or inconvenient to separate the first polynucleotide sequence from the second polynucleotide sequence or that the boundary between said first polynucleotide sequence and said second polynucleotide sequence is not known.
Thus, in either of the two above examples, when the method for the detection of the polynucleotide sequence of interest is carried out, the labeled second polynucleotide sequence is capable of hybridizing to a complementary polynucleotide sequence that may be contained in the sample, i.e. condition (2) is present. This can generate a false po itive result.